DNA which encodes trehalase and uses thereof

ABSTRACT

Disclosed are a DNA which encodes murine trehalase, a polypeptide expressed by the DNA, and a transgenic- and knockout-animals which have been genetically engineered with the DNA. The DNA comprises a part or the whole of the nucleotide sequence of SEQ ID NO:1.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a novel DNA, more particularly,to a novel DNA which encodes trehalase, and uses thereof.

[0003] 2. Description of the Prior Art

[0004] Trehalose, a non-reducing disaccharide which consists of glucoseunits as constituent saccharide, is widely distributed in the naturalworld, for example, in bacteria, fungi, algae, insects, Crustacean, etc.In organisms such as insects which have a relatively-large amount oftrehalose in their bodies, trehalose would play an important role asenergy source and relate to the maintenance of physiological functionsuch as cold resistance. Mammals including humans have long beenutilizing trehalose widely from mushrooms, seaweeds, fermented foods,etc, as reported by Oku et al., in “Journal of The Japanese Society ForFood Science And Technology”, Vol. 45, No. 6, pp. 381-384 (1998). Asdisclosed in Japanese Patent Kokai Nos. 143,876/95 and 213,283/95applied for by the present applicant, the establishment of technologiesfor industrial-scale production of trehalose has more increased theinterest of trehalose in the maintenance and regulation of biologicalfunctions in mammals, and this results in energetic and continuousresearches in various fields.

[0005] Trehalase is an enzyme which specifically hydrolyzes theglucosidic bond in trehalose. Because of this substrate specificity, theenzyme may deeply correlate to the trehalose level in vivo in organismssuch as insects and relate to the regulation of their biologicalfunctions. Even in mammals with no significant amount of trehalose,trehalase is found in animals such as humans, mice and rats. Majorphysiological role of trehalase in mammals would be the hydrolysis ofexternally-ingested trehalose when the saccharide is digested andabsorbed by the mammals. It was reported that trehalase is commonlyfound in specific mammalian organs independently of the intake oftrehalose, and hence there still remains many unknown biological rolesof trehalase per se. As described above, the roles of mammaliantrehalase in the maintenance and regulation of their physiologicalfunctions have also been focused recently, along with the increasinginterest in trehalose.

[0006] Methods in a molecular biological manner are very useful forelucidating the physiological roles of specific enzymes or polypeptidesin living bodies. Mice are the animals commonly used widely as modelsfor elucidating the biological functions of mammals including humans.Thus, the techniques and analyses for murine trehalase in a molecularbiological manner would be particularly useful for elucidating thephysiological roles of mammalian trehalase. Any nucleotide sequence ofmurine trehalase, which is requisite for its molecular biologicalengineering, has not been elucidated, and any structure of the enzymeper se has not been disclosed. Urgently expected are as follows: Theelucidation of a nucleotide sequence of a DNA for murine trehalase, theestablishment of a DNA useful for engineering the enzyme in a molecularbiological manner, and uses thereof.

SUMMARY OF THE INVENTION

[0007] In view of the foregoing, the object of the present invention isto provide a DNA useful for engineering murine trehalase in a molecularbiological manner, and uses thereof.

[0008] To attain the above object, the present inventors widely screenedcDNAs for RNAs, collected from mice, to isolate a cDNA for murinetrehalase. As a result, the present inventors obtained from a murineintestine a cDNA which consists of about 2,000 base pairs (hereinafterabbreviated as “bp”) and expresses a polypeptide with trehalaseactivity. When compared with nucleotide sequences of conventionallyknown DNAs, the cDNA contained, as a coding sequence of the polypeptide,a nucleotide sequence of SEQ ID NO: 1 which was clearly different fromconventionally known DNAs. The present inventors also found the factthat a gene for the cDNA exists on a murine chromosome. Based on theseresults, they confirmed that the cDNA was for murine trehalase, and thenanalyzed murine genomic DNAs with reference to the nucleotide sequenceof the cDNA, elucidated the structure of a gene for murine trehalasewhich consists of a total length of about 20,000 bp and comprises thenucleotide sequence of SEQ ID NO: 1 and introns for splitting thenucleotide sequence, and isolated the gene as a genomic DNA. The presentinventors also confirmed that a part or the whole of the isolated DNAcan be arbitrarily used to engineer and analyze murine trehalase in amolecular biological manner. For example, such a DNA can beadvantageously used to prepare transformants suitable for producingpolypeptides used as murine trehalase standard specimens and as antigensfor preparing anti-murine trehalase antibodies, and to preparetransgenic animals and trehalase gene knockout animals. The presentinvention is based on these findings.

[0009] The present invention solves the above object by providing a DNAwhich comprises a part or the whole of the nucleotide sequence of SEQ IDNO: 1 that encodes trehalase, a polypeptide obtainable by the expressionof the DNA, a process for producing the polypeptide using the DNA, and atransgenic- and knockout-animals obtainable therewith.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to a novel DNA which encodestrehalase and uses thereof. The term “trehalase” as referred to in thepresent invention means an enzyme, i.e., a protein or polypeptide whichspecially hydrolyzes the glucosidic bond in α,α-trehalose, and thehydrolysis activity is called “trehalase activity” in the presentinvention. The DNA of the present invention comprises a part or thewhole of the nucleotide sequence of SEQ ID NO: 1 which encodestrehalase, and usually it is mouse origin.

[0011] In the present invention, the term “a part or the whole of thenucleotide sequence of SEQ ID NO: 1” generally means “a nucleotidesequence which contains at least ten and several contiguous bases in thenucleotide sequence of SEQ ID NO: 1.”

[0012] Preferred examples of the DNA of the present invention includeDNAs as cDNAs which comprise a part or the whole of the nucleotidesequence of SEQ ID NO: 1, DNAs as genomic DNAs obtainable by fragmentingchromosomes, and other DNAs obtainable by applying replacement,deletion, and/or addition of bases to the above DNAs. These DNAs do notcontain telomeres as a characteristic structure in the terminal regionof chromosomes of eukaryotic cells such as mammalian cells, and suchDNAs are usually provided in the from of isolated liner DNAs which arecomposed of not more than about 20,000 bp and which are distinguishablefrom naturally-occurring chromosomal DNAs and RNAs present in mammaliancells. All of these DNAs according to the present invention may behomologous to the nucleotide sequences of cDNAs for human and rattrehalases, registered in “GenBank”, a database of nucleic acidsprovided by the National Institute of Health of USA, under the accessionNos. AB000824 and AF038043, respectively, but are not completelycoincided with the above registered nucleotide sequences.

[0013] A cDNA, as an example of the DNA of the present invention,usually comprises a part or the whole of the nucleotide sequence of SEQID NO: 1 as a coding sequence or nucleotide sequence which encodes thepolypeptide of the present invention, and may contain a nucleotidesequence as a non-coding region at the 5′ and/or 3′ end regions.Usually, such a cDNA can be obtained by preparing in the usual manner acDNA using a RNA as a template obtainable from organs such asintestines, kidneys, livers, and lungs from mice or their relativerodents, and cell lines established from these organs; and screening thecDNA with an index of the existence of annealing with at least a part ofthe nucleotide sequence of SEQ ID NO: 1. Any method such as PCR, colonyhybridization, or plaque hybridization method commonly used in this artcan be used as the screening method. Although the DNAs thus obtained arevaried depending on the nature of species, strains, individuals, andorgans as RNA sources, such DNAs are clearly homologous to thenucleotide sequence of SEQ ID NO: 1, usually they have a homology ofover 94%, more desirably, 97% or higher. For example, as described inthe later described examples, a DNA, which has the nucleotide sequenceof SEQ ID NO: 3 and includes the nucleotide sequence of SEQ ID NO: 1, isgenerally obtained by using RNAs from ddY murine intestines asmaterials.

[0014] A genomic DNA, as another form of the DNA of the presentinvention, generally contains a part or the whole of the nucleotidesequence of SEQ ID NO: 1 as a coding region, and another nucleotidesequence as an intron and non-translated region. For example, thegenomic DNA can be obtained by applying PCR using, as templates,chromosomes obtainable from appropriate organs and cells of mice andtheir relative rodents; and oligonucleotides as primers prepared basedon the nucleotide sequence of SEQ ID NO: 1; or by engineering genomiclibraries from the chromosomes in the usual manner, and screening thedesired DNAs using a probe prepared based on the nucleotide sequence ofSEQ ID NO: 1. Depending on the nature of species, strains, individuals,and organs of the sources of chromosomes, the DNAs thus obtained may bevaried in some degrees with respect to the nucleotide sequence of acoding sequence, non-translated region, and/or intron. The DNAs,however, have a clear homology to the nucleotide sequence of SEQ ID NO:1, usually, a homology of over 94%, more preferably, 97% or higher. Forexample, a chromosome from ICR Swiss mouse provides a DNA whichcomprises both the nucleotide sequence of SEQ ID NO: 1 and at least anintron that exists between the bases 80 and 81 in the nucleotidesequence. From a chromosome of C57BL/6 mouse, the following DNA can beobtained: A DNA which comprises the nucleotide sequence of SEQ ID NO: 1and at least the introns which exist respectively between the bases 181and 182, 326 and 327, 414 and 415, 515 and 516, 608 and 609, 725 and726, 848 and 849, 898 and 899, 1093 and 1094, 1311 and 1312, 1423 and1424, 1536 and 1537, and 1590 and 1591 in the nucleotide sequence of SEQID NO: 1. The above genomic DNA according to the present invention canbe usually obtained as a liner DNA which consists of about 20,000 basesor fewer.

[0015] As described above, the isolated DNA of the present invention canbe made modifications such as fragmentation, replacement, deletion,and/or addition of bases by conventional methods used in this art. TheDNA of the present invention includes the aforesaid modified DNAs aslong as they contain at least a part of the nucleotide sequence of SEQID NO: 1. For example, single-stranded DNAs or oligonucleotides, whichconsist of at least ten and several contiguous bases in the sense oranti-sense strand of the above DNAs, are useful as PCR primers andhybridization probes to detect or amplify the DNA of the presentinvention. The oligonucleotides can also be used to detect and amplifytrehalase-related DNAs, which comprise a part of the nucleotide sequenceof SEQ ID NO: 1, other than those illustrated in the presentspecification. The oligonucleotides as PCR primers can be incorporatedwith other nucleotide sequences such as restriction-enzyme-recognizingsites, and modified by replacement, deletion, and/or addition of baseswith or without altering the amino acid sequence encoded by theoligonucleotides. The DNAs as PCR primers, which have been received withany of the above modifications, can be arbitrarily used to preparerecombinant DNAs as expression vectors for expressing trehalase and astargeting vectors for preparing knockout mice. These DNAs in the form ofoligonucleotides can be obtained by chemical-synthetic-methods which arecommonly used in this art.

[0016] By applying conventional PCR reaction to the above DNAs, thefollowing DNAs can be obtained: DNAs which consist essentially of atleast a part of the coding sequences for the above exemplified DNAs, forexample, those which consist of either the nucleotide sequence of SEQ IDNO: 1 or bases 58 to 1728 in the nucleotide sequence of SEQ ID NO: 1,and nucleotide sequences which are partial nucleotide sequences of SEQID NO: 1 and which correspond to amino acid sequences that consist of atleast ten contiguous amino acids. For example, by using the recombinantDNA technology, these DNAs can be arbitrarily used to produce murinetrehalase-related polypeptides used as standard specimens forqualitative- and quantitative-analyses of murine trehalase, and asantigens for preparing anti-murine trehalase antibodies. In the case ofexpressing the aforesaid DNAS, replacement, addition, and/or deletion ofbases can be further introduced into a part of the DNAs, depending onthe hosts used. Examples of such modifications are as follows:

[0017] (i) Replacement of a part of the bases of the DNAs with referenceto the frequency of codons used in host cells without altering theinherent amino acid sequences encoded by the DNAs;

[0018] (ii) Addition of initiation and termination condons to the DNAs;

[0019] (iii) Addition of nucleotide sequences recognizable by specificsubstances such as histidine tag to the N- or C-terminus of polypeptidesto be expressed;

[0020] (iv) Deletion of a part of the bases of the DNAs in the 5′ or 3′end region to increase the efficiency of DNA expression; and

[0021] (v) Insertion of one or more of the above introns into suitablesites in the DNAs.

[0022] These DNAs of the present invention comprise the nucleotidesequences which usually have a homology of over 94%, more preferably,97% or higher to the sequence which consists of at least about 30contiguous bases in the nucleotide sequence of SEQ ID NO: 1. The DNA ofthe present invention includes those in the form of a recombinant DNAand those introduced into appropriate host cells.

[0023] The present invention provides a polypeptide obtainable by theexpression of any of the above DNAs. The polypeptide of the presentinvention comprises an amino acid sequence which has a homology of 94%or higher, more preferably, 97% or higher to an amino acid sequencewhich consists of ten and several contiguous amino acids in the aminoacid sequence of SEQ ID NO: 2; and may have trehalase activity. Forexample, an amino acid sequence, which consists of about 20 amino acidsin the N-terminal region of the amino acid sequence of SEQ ID NO: 2, iscapable of functioning as signal peptide; and another animo acidsequence, which consists of amino acids 20 to 576 in the amino acidsequence where the signal peptide has been eliminated, can participatein the expression of trehalase activity. The polypeptide of the presentinvention can be obtained in a desired amount by the following processof the present invention, which comprises the steps of producing thepolypeptide from cells capable of producing the polypeptide of thepresent invention, i.e., a polypeptide which comprises at least a partof the amino acid sequence encoded by the nucleotide sequence of SEQ IDNO: 1: and collecting the produced polypeptide. Transformants, whichhave been introduced with any of the above DNAs that consist essentiallyof at least a part of the coding sequence, are particularly useful asthe cells used in the process of the present invention. Thesetransformants can be usually obtained by annealing any of the above DNAswith autonomously-replicable vectors into recombinant DNAs, andintroducing the DNAs into appropriate hosts. The autonomously-replicablevectors can be selected from conventional plasmid vectors such as pCDM8,pcDNAI/Amp, pcDL-SR α, BCMGSNeo, pSV2-neo, pSV-2gpt, pEF-BOS, pCEV4,pME18S, pKY4, pKK223-3, pVL1392, and pVL1393. In general, the replicablevectors contain appropriate nucleotide sequences, which allow the DNA ofthe present invention to express in each hosts, such as promoters,enhancers, replication origins, termination sites, and splicing- and/orselective-sequences. Any method conventionally used in this art can beselectively used to ligate the DNA of the present invention to the abovevectors. For example, addition of linkers or sequences for recognizingrestriction enzymes by PCR, and treatment of restriction enzymes orligases are all useful.

[0024] Any cells derived from microorganisms, plants, and animalsincluding vertebrates such as mammals and invertebrates such as insects,which are all commonly used in this art to prepare transformant cells,can be used as host cells to be introduced with the DNA of the presentinvention. To prepare the polypeptide of the present invention at alesser cost and in a relatively-high yield, microorganisms such asEscherichia coli and Bacillus subtilis, and cells from insects can bepreferably used as hosts. Eukaryotic cells from yeasts and animals canbe preferably used when the polypeptide is directed to use in the fieldof reagents for researches or pharmaceuticals for mammals which requirea polypeptide equivalent to the one present naturally in the murine bodywith respect to saccharide chains to be added to and intra- andextra-cellar locations of the polypeptide. Examples of such host cellsfrom animals include COS-1 cells (ATCC CRL-1650), CHO-K1 cells (ATCCCCL-61), 3T3-Swiss albino cells (ATCC CCL-92), C127I cells (ATCCCRL-1616), CV-1 cells (ATCC CCL-70), HeLa cells (ATCC CCL-2), MOP-8cells (ATCC CRL-1709), and variants thereof; epithelial cells fromhumans, monkeys, mice, and hamsters; stromal cells: hematopoietic cells;and cells from insects such as Sf9 cells, commercialized by BDPharMingen, 10975 Torreyana Road, San Diego, Calif. 92121, USA, and HighFive commercialized by Invitrogen BV, NV Leek, Netherlands. To introducethe DNA of the present invention into the above host cells, any of thefollowing conventional methods can be used; DEAE-dextran method, calciumphosphate method, electroporation, lipofection method, microinjectionmethod, and virus infection method using retrovirus, adenovirus, herpesvirus, or vaccinia virus. Desired clones can be selected from theresulting transformants with an index of the existence of an introducedDNA or the productivity of the polypeptide. With regard to the aforesaidrecombinant DNAs and transformants, materials and methods commonly usedin this art are described in detail in “Current Immuno-protocol inMolecular Biology”, chapters 1-9 and 15-16 (1996), edited by FrederickM. Ausubel et al., published by John Wiley and Sons Inc., New York,U.S.A.

[0025] The transformants thus obtained produce the polypeptide of thepresent invention intra- or extra-cellularly by culturing orproliferating in media under the conditions selected depending on thetype of host cells or the structure of vectors used to introduce the DNAof the present invention. Although, the produced polypeptide can be usedintact to suit to final use, it is usually purified before use. For thepurification, any conventional methods commonly used in this art can beused, for example, salting out, dialysis, filtration, concentration,fractional precipitation, ion-exchange chromatography, gel filtrationchromatography, absorption chromatography, isoelectric chromatography,hydrophobic chromatography, reverse phase chromatography, affinitychromatography, gel electrophoresis, and isoelectric electrophoresis.The polypeptide of the present invention, purified to a desired level,can be obtained by allowing the resulting fraction, which has beenpurified by the above purification methods, to examine and analyzeproperties such as amino acid sequence, molecular weight, and trehalaseactivity of the polypeptide; and collecting a fraction which exhibitsthe desired properties.

[0026] In this art, once a desired DNA is obtained, it can be commonlyintroduced into appropriate animals to obtain so called transgenicanimals. The present invention also provides transgenic animals byapplying conventional methods to the DNA of the present invention. Thetransgenic animals can be generally prepared by introducing the DNA,which comprises at least a part of the aforesaid coding sequence, intoappropriate vectors which are selected depending on the species of thehosts used, in combination with another desired nucleotide sequences forpromoters or enhancers, etc., if necessary; and introducing theresulting recombinant DNA into fertilized eggs or embryonic stem cellsof host animals by either conventional methods such as microinjection orinfection with viruses. The following animals can be advantageously usedas host animals in the present invention because they can be bredeasily: Rodents such as mice, rats, and hamsters which are usedfrequently as experimental animals; and other mammals such as goats,sheep, pigs, and cows which are commonly bred as domestic animals. Theobtained cells introduced with the DNAs are transplanted to uterines oruterus of female animals in pseudopregnancy, which are the same speciesas the cells. Transgenic animals introduced with the DNA of the presentinvention can be obtained by applying hybridization or PCR method tonewborns born by natural delivery or Cesarean section, and selectinganimals introduced with the DNA. With these methods, the productivity ofthe polypeptide with trehalase activity of the present invention can beimparted to a desired animal. The transgenic animals thus obtained canbe arbitrarily used in the production of the polypeptide of the presentinvention and also used as animal models for examining the in vivoinfluence of the polypeptide in living bodies, and used as animals forscreening therapeutic, prophylactic, and diagnostic agents for mammaliandiseases related to the excessive production of the polypeptide. Thetechniques for preparing transgenic animals as mentioned above aredescribed in detail in “New Genetic Engineering Handbook”, pp. 269-276(1996), edited by Masami MURAMATSU, Hiroto OKAYAMA, and MasashiYAMAMOTO, published by Yodo Co., Ltd., Tokyo, Japan.

[0027] In this field, once the gene structure of a desired gene isrevealed and a DNA which contains at least a part of the gene isisolated, animals with artificially destroyed genes, i.e., knockoutanimals can be generally obtained. A general preparation method of aknockout animal is described in the below with reference to a knockoutmouse:

[0028] (i) Preparing a vector (targeting vector) to destroy a desiredgene;

[0029] (ii) Introducing the targeting vector into murine embryonic stemcells (ES cells) with totipotency;

[0030] (iii) Selecting ES cells where the desired gene has beendestroyed by the introduced targeting vector;

[0031] (iv) Infecting the selected ES cells into a murine blastula,transplanting the murine blastula to an expedient mouse, and selecting achimaera mouse from newborns delivered from the expedient mouse;

[0032] (v) Inbreeding a male chimera mouse with a female wild-typemouse, and selecting a male and female heterozygotes from F1 micedelivered from the female mouse; and

[0033] (vi) Inbreeding the male and female heterozygotes, and selectinga homozygote (knockout mouse) from F2 mice delivered from the femalemouse.

[0034] As mentioned above, since the present invention discloses a DNAas a genomic DNA which corresponds to a trehalase gene that comprises apart or the whole of the nucleotide sequence of SEQ ID NO: 1, knockoutanimals with a destroyed trehalase gene can be prepared by using the DNAin such a form. The present invention also provides such knockoutanimals. In the preparation of knockout animals of the presentinvention, targeting vectors used in the above step (i) are prepared.The targeting vector used in the present invention is generally preparedby applying replacement, deletion, and/or addition of bases to codingsequences of genomic DNAs, which contain at least apart of thenucleotide sequence of SEQ ID NO: 1, to modify the DNAs so as not toencode a polypeptide with trehalase activity, and introducing themodified DNAs into autonomously-replicable vectors. To ease theselection of ES cells in the above step (iii), the targeting vectorshould preferably be introduced with a sequence as positive and/ornegative selective-markers. Concrete examples of the positive selectivemarkers include a neomycin resistant gene and a β-galactosidase gene,and those of the negative selective markers include aherpes-simplex-virus-thymidine-kinase gene and adiphtheritic-toxin-A-fragment gene. By using methods such aselectroporation, the resulting targeting vector is usually digested withan appropriate restriction enzyme for linearization, and then introducedinto animal's ES cells with a desired trehalase gene, usually, intomurine ES cells. When homologous recombination occurs in cells whichhave been introduced with the targeting vector, the trehalase geneinherent to the cells is destroyed by replacing with a nucleotidesequence which is from the targeting vector and free of encoding apolypeptide with trehalose activity. The desired ES cells can beselected by screening the products from the cells introduced with thevector by using conventional methods such as PCR method for confirmingthe intracellular homologous recombination (the above step (iii)). Withthe ES cells thus obtained, the knockout animals of the presentinvention can be obtained by treating the cells in accordance with theabove steps (iv) to (vi). The obtained knockout animals are specificallyuseful as models for examining the in vivo physiological role oftrehalase and as animals for screening therapeutic, prophylactic, anddiagnostic agents for animal diseases correlated to defection orincompletion of trehalase. The preparation method for knockout animalsis called gene targeting and reported in detail in “New GeneticEngineering Handbook”, pp. 277-283 (1996), edited by Masami MURAMATSU,Hiroto OKAYAMA, and Masashi YAMAMOTO, published by Yodo Co., Ltd.,Tokyo, Japan.

[0035] The preferred embodiments according to the present invention aredescribed in the below with reference to the following Examples, and canbe modified or diversified according to the state of the art. In view ofthe state, the present invention should not be limited to theseExamples.

EXAMPLE 1 DNA Encoding Trehalase EXAMPLE 1-1 Isolation of Partial cDNAfrom Murine Intestine

[0036] The intestines used in this example were prepared from butcheredfive-week-old ddY female mice whose cervical vertebrates had beendislocated. The intestines were cut lengthwisely, and the inwalls werewashed with a physiological saline. Thereafter, epidermal cells of theintestines were taken 1.1 g by wet weight in conventional manner, andthen soaked in 7.7 ml of a mixture (pH 7.0) consisting of 5 M guanidineisothiocyanate, 10 mM EDTA, 50 mM Tris-HCl (pH 7.0), and 8 w/v % of2-mercaptoethanol, disrupted by a homogenizer, and kept at 4° C. for 15hours to obtain a cell disruptant. According to a conventional manner,to 35-ml centrifugation tubes were added one milliliter aliquots of 100mM EDTA (pH 7.5) containing 5.7 M cesium chloride, and 10 ml of the celldisruptant was layered on each solution. These tubes were subjected toultracentrifugation at 25,000 rpm and at 20° C. for 20 hours to obtainprecipitates as RNA fractions. To 15-ml centrifugation tubes were addedthese RNA fractions and equal volumes of a mixture of chloroform andisobutanol (=4:1 by volume), and the tubes were shaken for five minutesand centrifuged at 15,000 rpm and at 4° C. for 10 minutes. Thesupernatants were mixed with 2.5-fold volumes of ethanol and cooled at−20° C. for two hours to precipitate all RNAs. The precipitates werewashed with 75 v/v % aqueous ethanol, and dissolved in 0.5 ml of steriledistilled water to obtain the RNAs, followed by treating the RNAs with“Oligotex-dT30 <SUPER>”, an oligo (dT) binding bead commercialized byTakara Shuzo Co., Ltd., Otsu, Shiga, Japan, to collect and purify Poly(A)⁺ RNA. Thus, a mRNA of murine intestine in a solution form wasobtained.

[0037] An aqueous solution containing 1 μl of the mRNA was placed in a0.5-ml reaction tube, and then incubated at 70° C. for five minutes andcooled to 4° C. The resulting solution was mixed with 2 μof a 10×RTreaction buffer (an aqueous solution consisting of 200 mM Tris-HCl (pH8.0) and one mole per liter of potassium chloride), 2 μl of 25 mMmagnesium chloride, 2 μl of 100 mM dithiothreitol, 1 μl of 2.5 mM ofdNTPs, 1 μl of 0.2 μg/μl random hexamer, 0.5 μl of 35 units/μl “RNasin”,a ribonuclease inhibitor commercialized by PROMEGA Co., Ltd., Wisconsin,U.S.A., and 1 μl of 200 units/μl Moloney Murine Leukemia Virus(hereinafter abbreviated as “MMLV”). The mixture solution was brought upto a volume of 20 μl with sterile distilled water, and successivelyincubated at 25° C. for 10 minutes and at 42° C. for 30 minutes to forma first strand cDNA, followed the incubation at 99° C. for 5 minutes toterminate the reaction.

[0038] According to a conventional manner, a consensus sequence wasobtained by comparing cDNA sequences of human and rat trehalases,registered under the accession Nos. AB000824 and AF038043, respectively,in “GenBank”, a database of nucleic acids provided by the NationalInstitute of Health of USA. The oligonucleotide sequences of SEQ ID NO:4 and SEQ ID NO: 5 (hereinafter abbreviated as “s1” and “a1”,respectively), which had been designed based on the these results, werechemically synthesized in a conventional manner. To a reaction tube,containing the above first strand cDNA solution as a template, wereadded 5 μl of 10×Pfu reaction buffer, 1 μl of 2.5 units/μl of a Pfupolymerase commercialized by STRATAGENE Co., Ltd., California, U.S.A., 4μl of 2.5 mM dNTPs, 1 μl of 100 ng/μl oligonucleotide s1 as senseprimer, and 1 μl of 100 ng/μl oligonucleotide al as an antisense primer.The mixture solution was brought up to a volume of 50 μl with steriledistilled water. The mixture was subjected to a PCR reaction of 35cycles of successive incubations of at 94° C. for 45 seconds, 52° C. for45 seconds, and 72° C. for 3.5 minutes.

[0039] The PCR reaction product was electrophoresed on 1.2 w/v % agarosegel to give a main band of a DNA of about 1.5 kb. The DNA was extractedand purified on “QIAEX II Gel Extraction Kit”, a gel extraction kitcommercialized by QIAGEN Co., Ltd., Tokyo, Japan, according to theattached specification.

[0040] The purified DNA was ligated to “pCR-Script Amp SK (+)”, aplasmid vector, by using “pCR-Script SK (+) Cloning Kit”, a cloning kitcommercialized by STRATAGENE Co., Ltd., California, U.S.A., according tothe attached specification. A part of the ligated product was introducedinto “Epicurian Coli XL1-Blue MRF′ Kan supercompetent cells”, E. colicompetent cells attached to the kit, to transform the cells. Anappropriate amount of the transformed cells was inoculated to an LB agarplate containing 0.04 mg/ml X-gal and 4 μM of isopropyl thiogalactoside,and the plate was incubated at 37° C. for 16 hours. The formed colonieswere respectively suspended in 15 μl portions of sterile distilledwater, and the suspensions were heated at 95° C. for 5 minutes and thenpromptly cooled to 4° C. As mentioned above, the cooled suspensions astemplates were subjected to PCR, and the resulting PCR products weresubjected to agarose gel electrophoresis. A colony with a DNA of about1,500 bp was selected and inoculated into 2 ml of LB broth, followed bythe incubation at 37° C. for 16 hours under shaking conditions.

[0041] A recombinant DNA was extracted from the culture by the usualalkali-SDS method. The DNA was subjected to the usual dideoxy method toanalyze a nucleotide sequence and revealed that it contained thenucleotide sequence of SEQ ID NO: 6. The nucleotide sequence washomologous to but not clearly identical to nucleotide sequences of cDNAsof human and rat trehalases. Based on this, the recombinant DNA wasconfirmed to be a cDNA from murine intestine and named “pCRMTHa”.

EXAMPLE 1-2 Isolation of a Partial cDNA from Murine Intestine

[0042] The cDNA obtained in Example 1-1 was subjected to 5′RACE, amodified method of PCR, using “5′/3′ RACE Kit” commercialized by RocheDiagnostics Co., Ltd., Tokyo, Japan, and this confirmed the existence ofa nucleotide sequence which corresponded to the upstream of the5′-terminal region of the cDNA, followed by isolating a part of thecDNA. Oligonucleotides of SEQ ID Nos. 7 to 9 (hereinafter abbreviated as“a2”, “a3”, and “a4”, respectively) having nucleotide sequencescomplementary to inner nucleotide sequences in SEQ ID NO: 6, revealed inExample 1-1, were in a conventional manner chemically synthesized foruse as antisense primers in this method.

[0043] To a 0.5-ml reaction tube, containing 240 ng of the mRNA frommurine intestine obtained in Example 1-1, were added 4 μl of a 5×cDNAsynthesis solution (an aqueous solution consisting of 250 mMTris-HCl (pH8.5), 40 mM magnesium chloride, 150 mM potassium chloride, and 5 mMdithiothreitol), 2 μl of 2.5 mM dNTPs, 1 μl of 91 ng/μl oligonucleotidea2, and 1 μl of 20 units/μl reverse transcriptase from AvianMyeloblastosis Virus (hereinafter abbreviated as “AMV”). The mixturesolution was brought up to a volume of 20 μl with sterile distilledwater. The mixture was incubated at 55° C. for one hour and at 65° C.for 10 minutes to form a first strand cDNA. The reaction mixture wasadmixed with 100 μl of a reaction buffer consisting of 3M guanidinethiocyanate, 10 mMTris-HCl (pH6.6), and 5% ethanol. The resultingmixture was transferred to a spin column attached to the above kit, andthe spin column was centrifuged at 15,000 rpm for 30 seconds. To thespin column was added 500 μl of a washing buffer containing 20 mM sodiumchloride and 2 mM Tris-HCl (pH 7.5) in ethanol, and the contents in thecolumn were washed by centrifuging at 15,000 rpm for 30 seconds. Thecontents were rewashed with 200 μl of the same washing buffer bycentrifugation at 15,000 rpm for 30 seconds. To the column was added 50μl of an extraction buffer consisting of 10 mM Tris-HCl (pH 8.5) and 1mM EDTA, and the column was centrifuged at 15,000 rpm for 30 seconds,followed by collecting the extract to obtain a purified first strandcDNA.

[0044] To a 0.5-ml reaction tube, containing 19 μl of the above purifiedfirst strand cDNAs, were added 2.5 μl of 2 mM dATP and 2.5 μl of a10×reaction buffer consisting of 100 mM Tris-HCl (pH 8.3), 15 mMmagnesium chloride, and 500 mM potassium chloride in distilled water.The mixture was heated at 94° C. for 3 minutes, cooled, admixed with 1μl of 10 units/μl of a terminal transferase, and successively incubatedat 37° C. for 30 minutes and at 72° C. for 10 minutes, followed bybinding an oligo-dA to the 3′-terminus of the first strand cDNA. Fivemicroliters of the mixture were transferred to another reaction tube,admixed with 1 μl of 37.5 μM of an oligo-dT anchor primer attached tothe kit, 1 μl of 87 ng/μl oligonucleotide a3, 1 μl of 25 mM dNTPs, 1 μlof 2.5 units/μl Pfu polymerase, and 5 μl of a 10×Pfu reaction buffer,and the mixture solution was brought up to a volume of 50 μl withsterile distilled water. The resulting mixture was subjected to a firststep PCR of 35 cycles of successive incubations at 94° C. for 45seconds, 55° C. for 45 seconds, and 72° C. for two minutes.

[0045] One microliter of the product in the first step PCR wastransferred to another reaction tube, and then admixed with 1 μl of 12.5μM of a PCR anchor primer, 1 μl of 90 ng/μl oligonucleotide a4, dNTPs,Pfu polymerase, and Pfu reaction buffer similarly as in the first stepPCR. The mixture solution was subjected to a second step PCR whilecontrolling temperature similarly as in the first step PCR.

[0046] The second step PCR product was electrophoresed on 1.2 w/v %agarose gel in a conventional manner to give a main band of a DNA ofabout 550 bp. The DNA was extracted and purified on the gel extractionkit similarly as in Example 1-1. In accordance with Example 1-1, thepurified DNA was ligated to “pCR-Script Amp SK (+)”, a plasmid vector.The ligated product was introduced into E. coli, and the recombinant DNAhaving the desired DNA of about 550 bp was extracted from thetransformed cells. The recombinant DNA thus obtained was subjected tothe usual dideoxy method and revealed that it contained the nucleotidesequence in SEQ ID NO: 10. The sequence, consisting of 358 bases fromafter the base 182 in the 3′-terminal region of SEQ ID No: 10,completely coincided with a nucleotide sequence that consisted of 358bases in the 5′-terminal region of SEQ ID NO: 6, which had been revealedin Example 1-1. Based on this, it was confirmed that the recombinant DNAobtained in this example contained the desired partial cDNA from murineintestine. The recombinant DNA was named “pCRMTHb”.

EXAMPLE 1-3 Isolation of a Partial cDNA from Murine Intestine

[0047] To confirm the existence of a nucleotide sequence, correspondingto the 3′-terminal downstream region of the cDNA obtained in Example1-1, and to isolate a cDNA for the nucleotide sequence, the cDNAobtained in Example 1-1 was subjected to 3′RACE, a modified method ofPCR, by using “5′/3′ RACE Kit” similarly as in Example 1-2. Anoligonucleotide consisting of the nucleotide sequence in SEQ ID NO: 11(hereinafter abbreviated as “s2”), having a nucleotide sequencecomplementary to an inner sequence in SEQ ID NO: 6 which had beenrevealed in Example 1-1, was in a conventional manner chemicallysynthesized as a sense primer used in this method.

[0048] To a 0.5-ml reaction tube, containing 240 ng of mRNA from murineintestine obtained in Example 1-1, were added 4 μl of 5×cDNA synthesisbuffer as used in Example 1-2, 2 μl of 2.5 mM dNTPs, 1 μl of 37.5 μM ofan oligo-dT anchor primer, and 1 μl of 20 units/μl of reversetranscriptase from AMV. The mixture solution was brought up to a volumeof 20 μl with sterile distilled water, and then successively incubatedat 55° C. for one hour and at 65° C. for 10 minutes to obtain a firststrand cDNA.

[0049] To a new reaction tube, containing 1 μl of the first strand cDNA,were added 1 μl of 12.5 μM PCR anchor primer, 1 μl of 82 ng/μloligonucleotide s2, 1 μl of 25 mM dNTPs, 1 μl of 2.5 units/μl Pfupolymerase, and 5 μl of 10×Pfu reaction buffer. The mixture solution wasbrought up to a volume of 50μl with sterile distilled water andsubjected to a PCR reaction of 35 cycles of successive incubations at94° C. for 45 seconds, 55° C. for 45 seconds, and 72° C. for twominutes.

[0050] The PCR product was electrophoresed on 1.2 w/v % agarose gel in aconventional manner to give a main band of DNA of about 750 bp. The DNAwas extracted and purified by using the gel extraction similarly as inExample 1-1. In accordance with Example 1-1, the purified DNA wasligated to “pCR-Script Amp SK(+)”, a plasmid vector, and the ligatedproduct was introduced into E. coli. The recombinant DNA, whichcontained the desired DNA of about 750 bp, was extracted from thetransformed cells and subjected to the usual dideoxy method, revealingthat the DNA contained the nucleotide sequence of SEQ ID NO: 12. Anucleotide sequence, which consisted of 258 bases in the 5′-terminalregion in the above nucleotide sequence, completely corresponded to anucleotide sequence which consisted of 258 bases from after the base1214 in the 3′-terminal region of SEQ ID NO: 6 which had been revealedin Example 1-1. This revealed that the recombinant DNA in this examplecontained the desired partial cDNA from murine intestine. Therecombinant DNA was named “pCRMTHc”.

EXAMPLE 1-4 Preparation of a Full-Length cDNA from Murine Intestine

[0051] pCRMTHa, a recombinant DNA obtained in Example 1-1, hadrestriction sites of restriction enzymes EcoRI and AccI in thenucleotide sequence of cDNA from murine intestine, and also containedthese restriction sites within the nucleotide sequence of the plasmidvector in the downstream of the 3′-terminus. pCRMTHb, a recombinant DNAobtained in Example 1-2, had two restriction sites of EcoRI whichcorresponded to pCRMTHa. pCRMTHc, a recombinant DNA obtained in Example1-3, had two restriction sites of AccI which corresponded to pCRMTHa.With these structures, a recombinant DNA, having a full-length of cDNAobtained by linking together the above three types of partial cDNAs, wasprepared as indicated below:

[0052] About 2.4 μg of the pCRMTHa in Example 1-1 and about 2.4 μg ofthe pCRMTHb in Example 1-2 were in a conventional manner cleaved with asufficient amount of a restriction enzyme EcoRI. The digested productswere respectively electrophoresed on 1.2 w/v % agarose gel to give bandsfor DNAs of about 1,150 bp from pCRMTHa and of about 3,500 bp frompCRMTHb. These DNAs were purified after extracted from the agarose gelsusing a gel extraction kit similarly as in Example 1-1. These twopurified DNAs were ligated together with “DNA Ligation Kit version 2”, aligation kit commercialized by Takara Shuzo Co., Ltd., Tokyo, Japan. Inaccordance with the method in Example 1-1, E. coli were transformed withthe ligated product. A recombinant DNA having a DNA of about 1,650 bpwas extracted from the transformant and named “pCRMTHab”.

[0053] About 0.8 μg of the pCRMTHa obtained in the above and about 0.8μg of the pCRMTHc in Example 1-3 were respectively cleaved in aconventional manner with a sufficient amount of a restriction enzymeAccI. The resulting cleaved products were respectively electrophoresedon 1.2 w/v % agarose gel. DNAs of about 4,500 bp from pCRMTHab and about600 bp from pCRMTHc, which had been separated on the agarose gels, werepurified after extracted from the gels using a gel extraction kitsimilarly as in Example 1-1. These two purified DNAs were ligated usinga ligation kit as mentioned above. In accordance with Example 1-1, E.coli were transformed with the ligated product. A recombinant DNA havinga DNA of about 2,050 bp was extracted from a transformant and named“pCRMTHabc”.

[0054] The recombinant DNA was subjected to the usual dideoxy method toanalyze the nucleotide sequence and revealed that the DNA contained thenucleotide sequence of SEQ ID NO: 3. This nucleotide sequence containedall SEQ ID NOs: 6, 10, and 12, and this confirmed that pCRMTHabc, arecombinant DNA, included the desired ligated cDNA. Thus, a full-lengthcDNA from murine intestine was obtained. The nucleotide sequence,revealed in this example, encoded an amino acid sequence consisting of576 amino acids. A coding sequence for the nucleotide sequence of SEQ IDNO: 3 is separately shown in SEQ ID NO: 1.

EXAMPLE 1-5 Production of Polypeptides by Transformants EXAMPLE 1-5(a)Preparation of Recombinant DNA

[0055] A recombinant DNA for expressing polypeptide was prepared by aPCR method as shown below: The following oligonucleotides were firstchemically synthesized; an oligonucleotide in the nucleotide sequence ofSEQ ID NO: 13 (hereinafter abbreviated as “s3”), which had beenconstructed to include nucleotide sequences at the 5′-terminus in SEQ IDNO: 1 and a restriction site by a restriction enzyme of XhoI; and anoligonucleotide in the nucleotide sequence of SEQ ID NO: 14 (hereinafterabbreviated as “a5”), which had been constructed to include nucleotidesequences at the 3′-terminus in SEQ ID NO: 1 and a restriction site by arestriction enzyme of NotI.

[0056] To a 0.5-ml reaction tube, containing 2 ng of the recombinant DNA“pCRMTHabc” in Example 1-4, were added 10 μl of 10×Pfu reaction buffer,1 μl of 2.5 units/μl Pfu polymerase, 1 μl of 25 mM dNTPs, and adequateamounts of oligonucleotides s3 and a5. The mixture solution was broughtup to a volume of 100 μl with sterile distilled water, followed bysubjecting to a PCR reaction of 40 cycles of successive incubations at94° C. for 0.5 minutes, 54° C. for 2 minutes, and 72° C. for 3.5minutes. The resulting PCR product was ligated to “pCR-Script CamSK(+)”, a plasmid vector, by using “pCR-Script Cam SK(+) cloning kit”, acloning kit commercialized by STRATAGENE Co., Ltd., California, U.S.A.,according to the attached specification. By using the ligated product,E. coli JM109 competent cells, commercialized by Takara Shuzo Co.,Ltd.,Tokyo, Japan, were transformed in a conventional manner. The transformedcells were inoculated on LB agar plate (pH 7.2) containing 34 μg/mlchloramphenicol, and the plate was incubated at 37° C. for 18 hours. Aformed colony was inoculated into 2 ml of a liquid medium with similarcomposition as above and incubated at 37° C. for 18 hours under shakingconditions. A recombinant DNA was extracted from the culture by theusual alkali-SDS method. The dideoxy method confirmed that therecombinant DNA contained the nucleotide sequence of SEQ ID NO: 1 and astop codon at the 3′-terminus.

[0057] The recombinant DNA thus obtained was in a conventional mannercleaved with restriction enzymes of XhoI and NotI. pCDM8, a plasmidvector commercialized by Invitrogen BV, NV Leek, Netherlands, wassimilarly cleaved with the above restriction enzymes. These cleavedproducts were ligated by using “DNA ligation kit version 2”, a ligationkit, similarly as in Example 1-4. With the ligated products, E. coliMC1061/P3 competent cells, commercialized by Invitrogen BV, NV Leek,Netherlands, were transformed in a conventional manner. The transformedcells were inoculated on LB agar plate (pH 7.2) containing 20 μg/mlampicillin and 10 μg/ml tetracycline, and the plate was incubated 37° C.for 18 hours. The formed colony was inoculated into 2 ml of a liquidmedia with similar composition as above and incubated at 37° C. for 18hours under shaking conditions. A recombinant DNA was extracted from theculture by the usual alkali-SDS method. In the recombinant DNA thusobtained, the DNA of the nucleotide sequence of SEQ ID NO: 1 and a stopcodon were ligated in this order to the downstream of a promoter. Therecombinant DNA was named “pCDMTH”.

EXAMPLE 1-5(b) Confirmation of Production of Polypeptide and TrehalaseActivity

[0058] After COS-1 cells (ATCC CRL-1650), a kind of fibroblast celllines from a kidney of Africa green monkey, were in a conventionalmanner proliferated to give a prescribed cell concentration, theproliferated cells were transformed by introducing a recombinant DNA,prepared in Example 1-5(a), into the cells in an amount of 20 μg of“pCDMTH” per 7.5×10⁶ cells by the usual electroporation method. To aflat culture flask was added “Dulbecco's Modified Eagle Medium (Nissui2)”, commercialized by Nissui Pharmaceutical Co., Ltd., Tokyo, Japan,supplemented with 10 w/w % FCS. The transformed COS-1 cells wereinoculated to the medium at a rate of 2×10⁵ cells/ml, followed by theincubation at 37° C. for three days in a 5 v/v % CO₂ incubator in theusual manner. The culture was in a conventional manner separated into afraction of cells and a fraction of culture supernatant. The cells werewashed by repeating the following procedure for several times: Thefraction of cells was suspended in 10 mM sodium phosphate buffer (pH6.2) and centrifuged at 1,000 rpm and at 4° C. for five minutes,followed by removing the buffer. The fraction of washed cells wasadmixed with 4% triton X-100 and kept at ambient temperature for 10minutes. The fraction was centrifuged at 15,000 rpm and at 4° C. forfive minutes, followed by collecting a supernatant.

[0059] A trehalase activity of the supernatant was examined as follows:The obtained fraction was diluted with 10 mM sodium phosphate buffer (pH6.2) to give a desired concentration and mixed with α,α-trehalose togive a final concentration of 20 mM. Thereafter, the mixture wasincubated at 37° C. for one hour and mixed with 8 v/v % perchloric acid{fraction (1/10)} volume of the reaction mixture to suspend thereaction. Glucose in the mixture was quantified on “GLUCOSE B-TESTWAKO”,a kit for quantifying glucose commercialized by Wako Pure ChemicalIndustries, Ltd., Osaka, Japan. The amount of α,α-trehalose hydrolyzedin the reaction mixture was calculated based on the change in glucoselevel before and after the reaction. One unit trehalase activity wasdefined as an activity of hydrolyzing one μmol of α,α-trehalose perminute. The fraction thus obtained had 2.7 units of trehalase activity.When the above culture supernatant was similarly examined, significanttrehalase activity was not detected as found in the supernatant fractiontreated with triton X-100. In the case of operating a plasmid vector“pCDM8” instead of the recombinant DNA “pCDMTH” similarly as in thisexample, no trehalase activity was detected in any fraction.

[0060] The results in Examples 1-1 to 1-5 indicate that the nucleotidesequence of SEQ ID NO: 1, contained in “pCDMTH” obtained in thisexample, encodes a murine trehalase, and this means that the nucleotidesequence is usually contained in a murine trehalase gene. The nucleotidesequence of SEQ ID NO: 1 was compared with other conventional cDNAnucleotide sequences of trehalases with respect to their coincidedbases, and this revealed that the nucleotide sequence of the presentinvention had a highest homology of 94.0% to a cDNA of rat trehalase.The result shows that trehalases of mice and their closely relatedrodents are encoded by nucleotide sequences with a homology of at least94.0% to SEQ ID NO: 1.

[0061] The result in this example indicates that the murine trehalase isusually expressed as a polypeptide which has an amino acid sequence asshown in parallel in SEQ ID NO: 1 or the amino acid sequence of SEQ IDNO: 2. When examined for amino acid sequence homology based on thenumber of homologous amino acids, the above amino acid sequence had ahomology of 93.5% to that of rat trehalase. Conventional analysisconfirmed that hydrophobic amino acids, which are general characteristicsignal peptides, were focused on a part that consisted of about 20 aminoacids in the N-terminal region of the above amino acid sequence. Thisindicates that the part of murine trehalase possibly functions as asignal peptide, and a polypeptide other than the part predominantlyrelates to the expression of trehalase activity.

EXAMPLE 2 Preparation of Polypeptide with Trehalase Activity

[0062] A recombinant DNA “pCDMTH” in Example 1-5(a) was transformed byintroducing into COS-1 cells by the method in Example 1-5(b). Thetransformed cells were cultured according to the method in Example1-5(b), and a fraction of supernatant was obtained from the culturetreated with triton X-100. With an index of trehalase activity, thefraction was purified by combining gel filtration chromatography,ion-exchange chromatography, and isoelectric electrophoresis.Accordingly, a polypeptide with trehalase activity was obtained in arelatively-high purity and a yield of 2 mg per liter of culture.

[0063] The polypeptide is useful as a standard specimen in thequalitative and quantitative analyses of murine trehalase, an antigenfor preparing anti-murine trehalase antibodies, and a material for theantigen as a peptide.

[0064] As described above, the present invention is based on theisolation and the structural analysis of both a cDNA, which encodesmurine trehalase, and a gene for the cDNA. A variety of DNAs, providedby the present invention, enable to engineer and analyze murinetrehalase in a molecular biological manner. For example, according tothe present invention, the analysis of trehalase in living bodies suchas humans and animals, where the biological roles have not yet beenconfirmed, can be conducted in a molecular biological manner by usingmice useful as animal model. Such an animal model can also be used inresearches for treatment, preventive, and diagnostic preparations fordiseases such as diseases of trehalose metabolism deficiency caused bydeletion or incompletion of trehalase. The DNA of the present inventionprovides a relatively-large amount of murine trehalase which is valuablein conducting the above analysis. Trehalase would be a main enzyme whichrelates to the hydrolysis and metabolism of trehalose in living bodies.Accordingly, the present invention would provide trehalase and importantinformation in elucidating unknown physiological functions of trehalosein the bodies of mammals and humans.

[0065] Thus, the present invention having these outstanding functionsand effects is a significant invention that would greatly contribute tothis art.

[0066] While what are at present considered to be the preferredembodiments of the invention have been described, it will be understoodthat various modifications may be made therein, and the appended claimsare intended to cover all such modifications as fall within the truespirits and scope of the invention.

1 14 1 1728 DNA mouse 1 atg acc tgg gag ctg cac ctg ctg ctt ctg ctg gggctg gga ctt agg 48 Met Thr Trp Glu Leu His Leu Leu Leu Leu Leu Gly LeuGly Leu Arg 5 10 15 tcc cag gag gcc ctg cca cca ccc tgt gag agc cag atctac tgc cat 96 Ser Gln Glu Ala Leu Pro Pro Pro Cys Glu Ser Gln Ile TyrCys His 20 25 30 gga gag ctc ctg cac caa gtt cag atg gcc cag ctc tac caagat gac 144 Gly Glu Leu Leu His Gln Val Gln Met Ala Gln Leu Tyr Gln AspAsp 35 40 45 aag cag ttt gtg gat atg tca ctg gcc aca tct cca gat gaa gtcctg 192 Lys Gln Phe Val Asp Met Ser Leu Ala Thr Ser Pro Asp Glu Val Leu50 55 60 cag aag ttc agt gag ctg gcc aca gtc cac aac cac agc atc ccc aag240 Gln Lys Phe Ser Glu Leu Ala Thr Val His Asn His Ser Ile Pro Lys 6570 75 80 gaa cag ctt cag gaa ttt gtc cag agt cac ttc cag ccc gtg ggg cag288 Glu Gln Leu Gln Glu Phe Val Gln Ser His Phe Gln Pro Val Gly Gln 8590 95 gag ctg cag tcc tgg acc cct gag gac tgg aag gac agc cct cag ttc336 Glu Leu Gln Ser Trp Thr Pro Glu Asp Trp Lys Asp Ser Pro Gln Phe 100105 110 ctg cag aag atc tcg gat gct aat ctg cgt gtc tgg gcg gag gag cta384 Leu Gln Lys Ile Ser Asp Ala Asn Leu Arg Val Trp Ala Glu Glu Leu 115120 125 cac aag atc tgg aaa aag ctg gga aag aag atg aaa gca gaa gtc ctc432 His Lys Ile Trp Lys Lys Leu Gly Lys Lys Met Lys Ala Glu Val Leu 130135 140 agc tac ccc gag agg tcc tcc cta atc tac tca aag cac ccc ttc att480 Ser Tyr Pro Glu Arg Ser Ser Leu Ile Tyr Ser Lys His Pro Phe Ile 145150 155 160 gtg ccc ggg ggg cgc ttt gtt gaa ttc tac tac tgg gac tcg tactgg 528 Val Pro Gly Gly Arg Phe Val Glu Phe Tyr Tyr Trp Asp Ser Tyr Trp165 170 175 gtg atg gaa ggc ctg ctt ctt tct gag atg gcc tca aca gtg aagggt 576 Val Met Glu Gly Leu Leu Leu Ser Glu Met Ala Ser Thr Val Lys Gly180 185 190 atg ctg cag aac ttt ctg gat ctg gtg aag acc tac gga cat atcccc 624 Met Leu Gln Asn Phe Leu Asp Leu Val Lys Thr Tyr Gly His Ile Pro195 200 205 aac ggt gga cgc ata tat tac ctg caa cgg agc cag ccc cca ctcctg 672 Asn Gly Gly Arg Ile Tyr Tyr Leu Gln Arg Ser Gln Pro Pro Leu Leu210 215 220 act ctc atg atg gat cga tat gta gct cat acc aag gat gtc gccttc 720 Thr Leu Met Met Asp Arg Tyr Val Ala His Thr Lys Asp Val Ala Phe225 230 235 240 ctt cag gag aat att ggg act cta gcc tct gaa ctg gac ttctgg act 768 Leu Gln Glu Asn Ile Gly Thr Leu Ala Ser Glu Leu Asp Phe TrpThr 245 250 255 gtg aac agg act gtc tct gta gtc tca gga gga caa agc tatgtc tta 816 Val Asn Arg Thr Val Ser Val Val Ser Gly Gly Gln Ser Tyr ValLeu 260 265 270 aat cgc tac tat gtc cct tat ggg gga ccc agg cca gag tcctac agg 864 Asn Arg Tyr Tyr Val Pro Tyr Gly Gly Pro Arg Pro Glu Ser TyrArg 275 280 285 aaa gac gca gaa ttg gca aac tct gtg cca gaa ggg gac cgagag act 912 Lys Asp Ala Glu Leu Ala Asn Ser Val Pro Glu Gly Asp Arg GluThr 290 295 300 ctg tgg gct gag ctc aag gct ggg gct gag tct ggc tgg gacttc tct 960 Leu Trp Ala Glu Leu Lys Ala Gly Ala Glu Ser Gly Trp Asp PheSer 305 310 315 320 tca cgc tgg ctt gtt gga ggc cca gac cct gat ttg ctcagc agc atc 1008 Ser Arg Trp Leu Val Gly Gly Pro Asp Pro Asp Leu Leu SerSer Ile 325 330 335 cga acc agc aaa atg gta ccc gct gat ctg aac gcg ttcctg tgc caa 1056 Arg Thr Ser Lys Met Val Pro Ala Asp Leu Asn Ala Phe LeuCys Gln 340 345 350 gca gag gaa ctg atg agt aac ttc tac tcc aga cta gggaac gac aca 1104 Ala Glu Glu Leu Met Ser Asn Phe Tyr Ser Arg Leu Gly AsnAsp Thr 355 360 365 gag gcc aca aag tac agg aac ctg cgg gcc cag cgc ttggcc gcc atg 1152 Glu Ala Thr Lys Tyr Arg Asn Leu Arg Ala Gln Arg Leu AlaAla Met 370 375 380 gaa gct gtc ctg tgg gac gag cag aag ggt gcc tgg tttgac tat gac 1200 Glu Ala Val Leu Trp Asp Glu Gln Lys Gly Ala Trp Phe AspTyr Asp 385 390 395 400 ttg gaa aag ggg aag aag aac ctg gag ttt tat ccctcc aac ctc tcc 1248 Leu Glu Lys Gly Lys Lys Asn Leu Glu Phe Tyr Pro SerAsn Leu Ser 405 410 415 cca ctt tgg gct ggc tgc ttc tca gac cct agt gttgct gac aag gct 1296 Pro Leu Trp Ala Gly Cys Phe Ser Asp Pro Ser Val AlaAsp Lys Ala 420 425 430 ctg aag tac ttg gag gac agc aag atc ttg acc taccaa tat gga atc 1344 Leu Lys Tyr Leu Glu Asp Ser Lys Ile Leu Thr Tyr GlnTyr Gly Ile 435 440 445 cca acc tct ctt cgt aac aca ggc cag cag tgg gacttc ccc aat gcc 1392 Pro Thr Ser Leu Arg Asn Thr Gly Gln Gln Trp Asp PhePro Asn Ala 450 455 460 tgg gcc cca ctg cag gac ctg gtc att aga ggt ttggcc aag tca gct 1440 Trp Ala Pro Leu Gln Asp Leu Val Ile Arg Gly Leu AlaLys Ser Ala 465 470 475 480 tcc ccc cgg act cag gag gtg gct ttc cag ctggcc cag aat tgg atc 1488 Ser Pro Arg Thr Gln Glu Val Ala Phe Gln Leu AlaGln Asn Trp Ile 485 490 495 aaa acc aac ttc aaa gtc tac tcc caa aag tcagcg atg ttt gag aag 1536 Lys Thr Asn Phe Lys Val Tyr Ser Gln Lys Ser AlaMet Phe Glu Lys 500 505 510 tat gac atc agc aac ggt gga cat cca ggt ggagga ggg gag tat gaa 1584 Tyr Asp Ile Ser Asn Gly Gly His Pro Gly Gly GlyGly Glu Tyr Glu 515 520 525 gtt cag gaa gga ttt ggc tgg aca aac gga ttggcc ctg atg ctt ctg 1632 Val Gln Glu Gly Phe Gly Trp Thr Asn Gly Leu AlaLeu Met Leu Leu 530 535 540 gat cgc tat ggt gac cag ttg act tca ggg acccag tta gct tcc ctg 1680 Asp Arg Tyr Gly Asp Gln Leu Thr Ser Gly Thr GlnLeu Ala Ser Leu 545 550 555 560 gga ccc cac tgc cta gtg gct gcc ctt cttctc agt ctt ctg cta cag 1728 Gly Pro His Cys Leu Val Ala Ala Leu Leu LeuSer Leu Leu Leu Gln 565 570 575 2 576 PRT mouse 2 Met Thr Trp Glu LeuHis Leu Leu Leu Leu Leu Gly Leu Gly Leu Arg 5 10 15 Ser Gln Glu Ala LeuPro Pro Pro Cys Glu Ser Gln Ile Tyr Cys His 20 25 30 Gly Glu Leu Leu HisGln Val Gln Met Ala Gln Leu Tyr Gln Asp Asp 35 40 45 Lys Gln Phe Val AspMet Ser Leu Ala Thr Ser Pro Asp Glu Val Leu 50 55 60 Gln Lys Phe Ser GluLeu Ala Thr Val His Asn His Ser Ile Pro Lys 65 70 75 80 Glu Gln Leu GlnGlu Phe Val Gln Ser His Phe Gln Pro Val Gly Gln 85 90 95 Glu Leu Gln SerTrp Thr Pro Glu Asp Trp Lys Asp Ser Pro Gln Phe 100 105 110 Leu Gln LysIle Ser Asp Ala Asn Leu Arg Val Trp Ala Glu Glu Leu 115 120 125 His LysIle Trp Lys Lys Leu Gly Lys Lys Met Lys Ala Glu Val Leu 130 135 140 SerTyr Pro Glu Arg Ser Ser Leu Ile Tyr Ser Lys His Pro Phe Ile 145 150 155160 Val Pro Gly Gly Arg Phe Val Glu Phe Tyr Tyr Trp Asp Ser Tyr Trp 165170 175 Val Met Glu Gly Leu Leu Leu Ser Glu Met Ala Ser Thr Val Lys Gly180 185 190 Met Leu Gln Asn Phe Leu Asp Leu Val Lys Thr Tyr Gly His IlePro 195 200 205 Asn Gly Gly Arg Ile Tyr Tyr Leu Gln Arg Ser Gln Pro ProLeu Leu 210 215 220 Thr Leu Met Met Asp Arg Tyr Val Ala His Thr Lys AspVal Ala Phe 225 230 235 240 Leu Gln Glu Asn Ile Gly Thr Leu Ala Ser GluLeu Asp Phe Trp Thr 245 250 255 Val Asn Arg Thr Val Ser Val Val Ser GlyGly Gln Ser Tyr Val Leu 260 265 270 Asn Arg Tyr Tyr Val Pro Tyr Gly GlyPro Arg Pro Glu Ser Tyr Arg 275 280 285 Lys Asp Ala Glu Leu Ala Asn SerVal Pro Glu Gly Asp Arg Glu Thr 290 295 300 Leu Trp Ala Glu Leu Lys AlaGly Ala Glu Ser Gly Trp Asp Phe Ser 305 310 315 320 Ser Arg Trp Leu ValGly Gly Pro Asp Pro Asp Leu Leu Ser Ser Ile 325 330 335 Arg Thr Ser LysMet Val Pro Ala Asp Leu Asn Ala Phe Leu Cys Gln 340 345 350 Ala Glu GluLeu Met Ser Asn Phe Tyr Ser Arg Leu Gly Asn Asp Thr 355 360 365 Glu AlaThr Lys Tyr Arg Asn Leu Arg Ala Gln Arg Leu Ala Ala Met 370 375 380 GluAla Val Leu Trp Asp Glu Gln Lys Gly Ala Trp Phe Asp Tyr Asp 385 390 395400 Leu Glu Lys Gly Lys Lys Asn Leu Glu Phe Tyr Pro Ser Asn Leu Ser 405410 415 Pro Leu Trp Ala Gly Cys Phe Ser Asp Pro Ser Val Ala Asp Lys Ala420 425 430 Leu Lys Tyr Leu Glu Asp Ser Lys Ile Leu Thr Tyr Gln Tyr GlyIle 435 440 445 Pro Thr Ser Leu Arg Asn Thr Gly Gln Gln Trp Asp Phe ProAsn Ala 450 455 460 Trp Ala Pro Leu Gln Asp Leu Val Ile Arg Gly Leu AlaLys Ser Ala 465 470 475 480 Ser Pro Arg Thr Gln Glu Val Ala Phe Gln LeuAla Gln Asn Trp Ile 485 490 495 Lys Thr Asn Phe Lys Val Tyr Ser Gln LysSer Ala Met Phe Glu Lys 500 505 510 Tyr Asp Ile Ser Asn Gly Gly His ProGly Gly Gly Gly Glu Tyr Glu 515 520 525 Val Gln Glu Gly Phe Gly Trp ThrAsn Gly Leu Ala Leu Met Leu Leu 530 535 540 Asp Arg Tyr Gly Asp Gln LeuThr Ser Gly Thr Gln Leu Ala Ser Leu 545 550 555 560 Gly Pro His Cys LeuVal Ala Ala Leu Leu Leu Ser Leu Leu Leu Gln 565 570 575 3 2045 DNA mouseCDS (24)...(1751) 3 ccgttctagg caccgtgccc agg atg acc tgg gag ctg cacctg ctg ctt ctg 53 Met Thr Trp Glu Leu His Leu Leu Leu Leu 5 10 ctg gggctg gga ctt agg tcc cag gag gcc ctg cca cca ccc tgt gag 101 Leu Gly LeuGly Leu Arg Ser Gln Glu Ala Leu Pro Pro Pro Cys Glu 15 20 25 agc cag atctac tgc cat gga gag ctc ctg cac caa gtt cag atg gcc 149 Ser Gln Ile TyrCys His Gly Glu Leu Leu His Gln Val Gln Met Ala 30 35 40 cag ctc tac caagat gac aag cag ttt gtg gat atg tca ctg gcc aca 197 Gln Leu Tyr Gln AspAsp Lys Gln Phe Val Asp Met Ser Leu Ala Thr 45 50 55 tct cca gat gaa gtcctg cag aag ttc agt gag ctg gcc aca gtc cac 245 Ser Pro Asp Glu Val LeuGln Lys Phe Ser Glu Leu Ala Thr Val His 60 65 70 aac cac agc atc ccc aaggaa cag ctt cag gaa ttt gtc cag agt cac 293 Asn His Ser Ile Pro Lys GluGln Leu Gln Glu Phe Val Gln Ser His 75 80 85 90 ttc cag ccc gtg ggg caggag ctg cag tcc tgg acc cct gag gac tgg 341 Phe Gln Pro Val Gly Gln GluLeu Gln Ser Trp Thr Pro Glu Asp Trp 95 100 105 aag gac agc cct cag ttcctg cag aag atc tcg gat gct aat ctg cgt 389 Lys Asp Ser Pro Gln Phe LeuGln Lys Ile Ser Asp Ala Asn Leu Arg 110 115 120 gtc tgg gcg gag gag ctacac aag atc tgg aaa aag ctg gga aag aag 437 Val Trp Ala Glu Glu Leu HisLys Ile Trp Lys Lys Leu Gly Lys Lys 125 130 135 atg aaa gca gaa gtc ctcagc tac ccc gag agg tcc tcc cta atc tac 485 Met Lys Ala Glu Val Leu SerTyr Pro Glu Arg Ser Ser Leu Ile Tyr 140 145 150 tca aag cac ccc ttc attgtg ccc ggg ggg cgc ttt gtt gaa ttc tac 533 Ser Lys His Pro Phe Ile ValPro Gly Gly Arg Phe Val Glu Phe Tyr 155 160 165 170 tac tgg gac tcg tactgg gtg atg gaa ggc ctg ctt ctt tct gag atg 581 Tyr Trp Asp Ser Tyr TrpVal Met Glu Gly Leu Leu Leu Ser Glu Met 175 180 185 gcc tca aca gtg aagggt atg ctg cag aac ttt ctg gat ctg gtg aag 629 Ala Ser Thr Val Lys GlyMet Leu Gln Asn Phe Leu Asp Leu Val Lys 190 195 200 acc tac gga cat atcccc aac ggt gga cgc ata tat tac ctg caa cgg 677 Thr Tyr Gly His Ile ProAsn Gly Gly Arg Ile Tyr Tyr Leu Gln Arg 205 210 215 agc cag ccc cca ctcctg act ctc atg atg gat cga tat gta gct cat 725 Ser Gln Pro Pro Leu LeuThr Leu Met Met Asp Arg Tyr Val Ala His 220 225 230 acc aag gat gtc gccttc ctt cag gag aat att ggg act cta gcc tct 773 Thr Lys Asp Val Ala PheLeu Gln Glu Asn Ile Gly Thr Leu Ala Ser 235 240 245 250 gaa ctg gac ttctgg act gtg aac agg act gtc tct gta gtc tca gga 821 Glu Leu Asp Phe TrpThr Val Asn Arg Thr Val Ser Val Val Ser Gly 255 260 265 gga caa agc tatgtc tta aat cgc tac tat gtc cct tat ggg gga ccc 869 Gly Gln Ser Tyr ValLeu Asn Arg Tyr Tyr Val Pro Tyr Gly Gly Pro 270 275 280 agg cca gag tcctac agg aaa gac gca gaa ttg gca aac tct gtg cca 917 Arg Pro Glu Ser TyrArg Lys Asp Ala Glu Leu Ala Asn Ser Val Pro 285 290 295 gaa ggg gac cgagag act ctg tgg gct gag ctc aag gct ggg gct gag 965 Glu Gly Asp Arg GluThr Leu Trp Ala Glu Leu Lys Ala Gly Ala Glu 300 305 310 tct ggc tgg gacttc tct tca cgc tgg ctt gtt gga ggc cca gac cct 1013 Ser Gly Trp Asp PheSer Ser Arg Trp Leu Val Gly Gly Pro Asp Pro 315 320 325 330 gat ttg ctcagc agc atc cga acc agc aaa atg gta ccc gct gat ctg 1061 Asp Leu Leu SerSer Ile Arg Thr Ser Lys Met Val Pro Ala Asp Leu 335 340 345 aac gcg ttcctg tgc caa gca gag gaa ctg atg agt aac ttc tac tcc 1109 Asn Ala Phe LeuCys Gln Ala Glu Glu Leu Met Ser Asn Phe Tyr Ser 350 355 360 aga cta gggaac gac aca gag gcc aca aag tac agg aac ctg cgg gcc 1157 Arg Leu Gly AsnAsp Thr Glu Ala Thr Lys Tyr Arg Asn Leu Arg Ala 365 370 375 cag cgc ttggcc gcc atg gaa gct gtc ctg tgg gac gag cag aag ggt 1205 Gln Arg Leu AlaAla Met Glu Ala Val Leu Trp Asp Glu Gln Lys Gly 380 385 390 gcc tgg tttgac tat gac ttg gaa aag ggg aag aag aac ctg gag ttt 1253 Ala Trp Phe AspTyr Asp Leu Glu Lys Gly Lys Lys Asn Leu Glu Phe 395 400 405 410 tat ccctcc aac ctc tcc cca ctt tgg gct ggc tgc ttc tca gac cct 1301 Tyr Pro SerAsn Leu Ser Pro Leu Trp Ala Gly Cys Phe Ser Asp Pro 415 420 425 agt gttgct gac aag gct ctg aag tac ttg gag gac agc aag atc ttg 1349 Ser Val AlaAsp Lys Ala Leu Lys Tyr Leu Glu Asp Ser Lys Ile Leu 430 435 440 acc taccaa tat gga atc cca acc tct ctt cgt aac aca ggc cag cag 1397 Thr Tyr GlnTyr Gly Ile Pro Thr Ser Leu Arg Asn Thr Gly Gln Gln 445 450 455 tgg gacttc ccc aat gcc tgg gcc cca ctg cag gac ctg gtc att aga 1445 Trp Asp PhePro Asn Ala Trp Ala Pro Leu Gln Asp Leu Val Ile Arg 460 465 470 ggt ttggcc aag tca gct tcc ccc cgg act cag gag gtg gct ttc cag 1493 Gly Leu AlaLys Ser Ala Ser Pro Arg Thr Gln Glu Val Ala Phe Gln 475 480 485 490 ctggcc cag aat tgg atc aaa acc aac ttc aaa gtc tac tcc caa aag 1541 Leu AlaGln Asn Trp Ile Lys Thr Asn Phe Lys Val Tyr Ser Gln Lys 495 500 505 tcagcg atg ttt gag aag tat gac atc agc aac ggt gga cat cca ggt 1589 Ser AlaMet Phe Glu Lys Tyr Asp Ile Ser Asn Gly Gly His Pro Gly 510 515 520 ggagga ggg gag tat gaa gtt cag gaa gga ttt ggc tgg aca aac gga 1637 Gly GlyGly Glu Tyr Glu Val Gln Glu Gly Phe Gly Trp Thr Asn Gly 525 530 535 ttggcc ctg atg ctt ctg gat cgc tat ggt gac cag ttg act tca ggg 1685 Leu AlaLeu Met Leu Leu Asp Arg Tyr Gly Asp Gln Leu Thr Ser Gly 540 545 550 acccag tta gct tcc ctg gga ccc cac tgc cta gtg gct gcc ctt ctt 1733 Thr GlnLeu Ala Ser Leu Gly Pro His Cys Leu Val Ala Ala Leu Leu 555 560 565 570ctc agt ctt ctg cta cag tgacaagaac aagaatggac tcactgcctg 1781 Leu SerLeu Leu Leu Gln 575 cgctttctcc cctggcccca gctcatggtt cattaaacccttgctctacc ttcccttata 1841 gccccacccc caccatgccc cttcctgctc ataatgtgtcctgagccaag aagtgaccaa 1901 gaggtcaaga ctgtaatttt cacagtgttc tgagccaagaagtgaccaag aggtcaaggt 1961 tgtaattttc acgagggcgg aaactgaatc ctgatacctaaagtatccta tcgggtacca 2021 aatatagctc agagttccac actc 2045 4 20 DNAArtificial Sequence Designed oligonucleotide based on conservednucleotide sequences in cDNAs for human and rat trehalase 4 gatgacaagcagtttgtgga 20 5 18 DNA Artificial Sequence Designed oligonucleotidebased on conserved nucleotide sequences in cDNAs for human and rattrehalase 5 gtcaccatag cggtccag 18 6 1471 DNA mouse 6 tatgtcactggccacatctc cagatgaagt cctgcagaag ttcagtgagc tggccacagt 60 ccacaaccacagcatcccca aggaacagct tcaggaattt gtccagagtc acttccagcc 120 cgtggggcaggagctgcagt cctggacccc tgaggactgg aaggacagcc ctcagttcct 180 gcagaagatctcggatgcta atctgcgtgt ctgggcggag gagctacaca agatctggaa 240 aaagctgggaaagaagatga aagcagaagt cctcagctac cccgagaggt cctccctaat 300 ctactcaaagcaccccttca ttgtgcccgg ggggcgcttt gttgaattct actactggga 360 ctcgtactgggtgatggaag gcctgcttct ttctgagatg gcctcaacag tgaagggtat 420 gctgcagaactttctggatc tggtgaagac ctacggacat atccccaacg gtggacgcat 480 atattacctgcaacggagcc agcccccact cctgactctc atgatggatc gatatgtagc 540 tcataccaaggatgtcgcct tccttcagga gaatattggg actctagcct ctgaactgga 600 cttctggactgtgaacagga ctgtctctgt agtctcagga ggacaaagct atgtcttaaa 660 tcgctactatgtcccttatg ggggacccag gccagagtcc tacaggaaag acgcagaatt 720 ggcaaactctgtgccagaag gggaccgaga gactctgtgg gctgagctca aggctggggc 780 tgagtctggctgggacttct cttcacgctg gcttgttgga ggcccagacc ctgatttgct 840 cagcagcatccgaaccagca aaatggtacc cgctgatctg aacgcgttcc tgtgccaagc 900 agaggaactgatgagtaact tctactccag actagggaac gacacagagg ccacaaagta 960 caggaacctgcgggcccagc gcttggccgc catggaagct gtcctgtggg acgagcagaa 1020 gggtgcctggtttgactatg acttggaaaa ggggaagaag aacctggagt tttatccctc 1080 caacctctccccactttggg ctggctgctt ctcagaccct agtgttgctg acaaggctct 1140 gaagtacttggaggacagca agatcttgac ctaccaatat ggaatcccaa cctctcttcg 1200 taacacaggccagcagtggg acttccccaa tgcctgggcc ccactgcagg acctggtcat 1260 tagaggtttggccaagtcag cttccccccg gactcaggag gtggctttcc agctggccca 1320 gaattggatcaaaaccaact tcaaagtcta ctcccaaaag tcagcgatgt ttgagaagta 1380 tgacatcagcaacggtggac atccaggtgg aggaggggag tatgaagttc aggaaggatt 1440 tggctggacaaacggattgg ccctgatgct t 1471 7 22 DNA Artificial Sequence Designedoligonucleotide to have complementary sequence to internal part of SEQID NO6 7 tatgtccgta gttcttcacc ag 22 8 21 DNA Artificial SequenceDesigned oligonucleotide to have complementary sequence to internal partof SEQ ID NO6 8 gcagcatacc cttcactgtt g 21 9 21 DNA Artificial SequenceDesigned oligonucleotide to have complementary sequence to internal partof SEQ ID NO6 9 ttccatcacc cagtacgagt c 21 10 539 DNA mouse 10ccgttctagg caccgtgccc aggatgacct gggagctgca cctgctgctt ctgctggggc 60tgggacttag gtcccaggag gccctgccac caccctgtga gagccagatc tactgccatg 120gagagctcct gcaccaagtt cagatggccc agctctacca agatgacaag cagtttgtgg 180atatgtcact ggccacatct ccagatgaag tcctgcagaa gttcagtgag ctggccacag 240tccacaacca cagcatcccc aaggaacagc ttcaggaatt tgtccagagt cacttccagc 300ccgtggggca ggagctgcag tcctggaccc ctgaggactg gaaggacagc cctcagttcc 360tgcagaagat ctcggatgct aatctgcgtg tctgggcgga ggagctacac aagatctgga 420aaaagctggg aaagaagatg aaagcagaag tcctcagcta ccccgagagg tcctccctaa 480tctactcaaa gcaccccttc attgtgcccg gggggcgctt tgttgaattc tactactgg 539 1120 DNA Artificial Sequence Designed oligonucleotide to have internalsequence of SEQ ID NO6 11 ttcttcgtaa cacaggccag 20 12 651 DNA mouse 12cagtgggact tccccaatgc ctgggcccca ctgcaggacc tggtcattag aggtttggcc 60aagtcagctt ccccccggac tcaggaggtg gctttccagc tggcccagaa ttggatcaaa 120accaacttca aagtctactc ccaaaagtca gcgatgtttg agaagtatga catcagcaac 180ggtggacatc caggtggagg aggggagtat gaagttcagg aaggatttgg ctggacaaac 240ggattggccc tgatgcttct ggatcgctat ggtgaccagt tgacttcagg gacccagtta 300gcttccctgg gaccccactg cctagtggct gcccttcttc tcagtcttct gctacagtga 360caagaacaag aatggactca ctgcctgcgc tttctcccct ggccccagct catggttcat 420taaacccttg ctctaccttc ccttatagcc ccacccccac catgcccctt cctgctcata 480atgtgtcctg agccaagaag tgaccaagag gtcaagactg taattttcac agtgttctga 540gccaagaagt gaccaagagg tcaaggttgt aattttcacg agggcggaaa ctgaatcctg 600atacctaaag tatcctatcg ggtaccaaat atagctcaga gttccacact c 651 13 28 DNAArtificial Sequence Designed oligonucleotide to contain 5′-terminalsequence of SEQ ID NO1 and Xho I recognition site 13 aatctcgagccaccatgacc tgggagct 28 14 29 DNA Artificial Sequence Designedoligonucleotide to contain complementary sequence to 3′-terminal of SEQID NO1 and Not I recognition si 14 tctgcggccg cttactgtag cagaagact 29

What is claimed is:
 1. A polypeptide which is obtainable by theexpression of a DNA which comprises the nucleotide sequence of SEQ IDNO:1 that encodes a trehalase or a fragment thereof.
 2. The polypeptideof claim 1, which has trehalase activity.
 3. The polypeptide of claim 1which contains the amino acid sequence of SEQ ID NO:2 or a fragmentthereof.
 4. A process for producing polypeptide, which comprises:allowing a transformed cell, which has been obtained by introducing aDNA, which comprises the nucleotide sequence of SEQ ID NO:1 that encodesa trehalase or a fragment thereof, into an appropriate host cell derivedfrom a microorganism, plant or animal including insect, to express apolypeptide encoded by the DNA; and collecting the expressedpolypeptide, wherein said polypeptide comprises a part or the whole ofthe amino acid sequence of SEQ ID NO:2.
 5. The process of claim 4,wherein said polypeptide has trehalase activity.
 6. The process of claim4, wherein said produced polypeptide is collected by one or morepurification methods selected from the group consisting of dialysis,salting out, filtration, concentration, fractional precipitation, gelfiltration chromatography, ion-exchange chromatography, hydrophobicchromatography, reverse phase chromatography, affinity chromatography,gel electrophoresis, and isoelectric electrophoresis.
 7. A transgenicanimal, into which a part or the whole of the nucleotide sequence of SEQID NO:1 has been introduced.
 8. A knockout animal, which a trehalasegene, comprising a part or the whole of the nucleotides sequence SEQ IDNO:1, has been destroyed.